Actin and myosin are involved in a host of contractile events in nonmuscle cells including cytokinesis, phagocytosis and cell locomotion. The regulation of the interaction of these two proteins is critical to normal cell function. There are at least two regulatory systems involved in this; one is myosin light chain kinase-dependent phosphorylation of myosin. This kinase is regulated by calcium and calmodulin. Myosin must be phosphorylated in order to be activated. The other system involves caldesmon, an actin and calmodulin binding protein which can inhibit myosin activity in vitro. The inhibition can be reversed by calmodulin binding to caldesmon. Intact platelet caldesmon and a carboxy-terminal fragment prepared by recombinant DNA techniques and expressed in E. coli inhibits the sliding of fluorescently labeled actin filaments over myosin bound to a surface in an in vitro motility assay while the amino-terminus of caldesmon does not. It has been shown that caldesmon is phosphorylated when human platelets are treated with Prostacyclin, a prostaglandin, at sites phosphorylated in vitro by cAMP-dependent (Sigma) protein kinase. There are three major sites of phosphorylation of caldesmon by cAMP-dependent protein kinase as have been shown by enzymatic digest of phosphorylated caldesmon and isoelectric focusing. By using a recombinant caldesmon fragment purified from E. coli, one of these sites was localized in the N-terminal and was found to be Ser 202. Very recently, our preliminary results have confirmed the above observation, using intact purified platelet caldesmon. We have also identified a second phosphorylation site of human platelet caldesmon: Ser 263, using Achromobacter protease I digest and HPLC. This site remains to be confirmed. In that pursuit, we have been using cyanogen bromide cleavage and PVDF membrane electroblotting to facilitate the sequencing of the phosphopeptide. As previously reported, we have noted 3 distinct bands on an autoradiogram with the lowest band being approximately 12 kD.